Trafficking of proteins across the nuclear envelope is fundamental to the function of all living cells. In cancer and endocrine disorders this highly regulated process may be disrupted. This proposal explores aspects of the molecular mechanisms regulating shuttling of thyroid hormone receptor (TR) subtypes, including TR?1, TR?1, TR?2, and the oncoprotein v-ErbA. The thyroid hormone receptors are essential transcription factors that either activate or repress the expression of target genes in response to thyroid hormone. From our prior studies, a rich and complex picture is emerging of gene expression modulated by a dynamic balance between TR nuclear import, nuclear retention, and nuclear export. In the proposed research, we will determine the relative contribution of altered intracellular distribution patterns of TR to pathogenesis. Our prior studies have shown that even single amino acid changes in TR dramatically alter intracellular distribution patterns. We reasoned that mutations within the regions housing nuclear localization signal (NLS) and nuclear export signal (NES) motifs, which affect nuclear shuttling activity, will prove to play an important role in some forms of human cancer or Resistance to Thyroid Hormone (RTH) syndrome. Here, we will determine whether mutations in TR?1 that are linked to cancer or RTH alter nuclear import or export activity, intranuclear mobility and retention, or sequestration in aggresomes and other cytosolic inclusions in distinct ways. Given that TR contains multiple, conflicting import and export signals, the question arises of what factors are involved in nuclear retention of TR. In the proposed research, we will investigate the role of Mediator subunits MED1 and MED13 in nuclear retention of TR. We will use our recent advances in understanding of TR's critical domains and a powerful combined approach of RNA interference (RNAi), fluorescence recovery after photobleaching (FRAP), and in situ nuclear retention assays to assess factors affecting intranuclear dynamics of TR variants. The interplay between Mediator, importins, exportins, and NLSs and NESs adds another relatively unexplored dimension to regulation of TR trafficking. Results of the proposed studies will probe deeper into how the fine balance of TR distribution is regulated, providing insight into the correlation between altered trafficking signals in TR and disease manifestation, and increasing understanding of the normal cellular response to thyroid hormone. Our analyses will build a comprehensive model for how multiple signals can interact to mediate transcription factor localization and function. Our overarching aspiration is to inform the development of new therapeutic strategies for TR-associated disorders.